PROJECT SUMMARY The goal of this project is to gain mechanistic understanding about a class of transmembrane proteins called calcium homeostasis modulator (CALHM) that form hemichannels for ions and substrates such as ATP. Studies in the last decade showed that CALHM proteins are involved in neurological processes associated with Alzheimer?s disease (AD) and depression. A single nucleotide polymorphism (SNP) within the calhm1 gene that results in the Pro86Leu mutation results in increased deposition of amyloid beta, a hallmark of AD, and likely modulates the age of AD onset by interacting with the effect of the ?4 allele of the ApoE gene. Knockout of CALHM1 in mice has been shown to significantly impair flexible memory by altering cerebral neuronal activity via differential phosphorylation of the two glutamate-gated ion channels, NMDA receptors and AMPA receptors, which are involved in neuronal plasticity. In addition, conditional knockout of CALHM2 in astrocyte has been shown to result in decreased spine density and long term potentiation as well as depression-like behavior, which can be rescued by ATP replenishment. Overall, CALHM proteins are generally involved in controlling excitability of neurons and dysfunctional CALHM can result in neurological disorders and diseases, implying that controlling the function of CALHM may be a relevant therapeutic strategy. However, despite playing critical roles in the brain physiology and pathology, molecular-based understanding of CALHM proteins has lagged behind as there is no structure of any member of CALHM proteins to date. Thus, the goal of the proposed project is to unravel the first structure of the CALHM proteins to gain mechanistic insight into physiological function and pharmacology of the CALHM family. We will achieve these goals by conducting research with the following aims: Aim 1 establishing the method for obtaining milligram quantity of CALHM proteins for biophysical assays and cell-based and the platform for liposome-based assays for effective assessment of CALHM functions; Aim 2 revealing the first structure of CALHM2 proteins in various functional states to understand how they form multi-oligomeric channels and how they are activated and inhibited; and Aim 3 unraveling the first structure of CALHM1 and the CALHM1 Pro86Leu in various functional states to understand the molecular basis for the functional differences between the WT and the mutant CALHM1 as well as between CALHM1 and 2. Aims 2-3 will be achieved by implementing cryo-electron microscopy (cryo-EM)/single particle analysis. The structures will be validated by biochemical method and structure-based functional hypotheses will be tested by assays established in Aim 1 including cell- based and liposome-based ATP flux assay and electrophysiology. Successful completion of the proposed studies will provide in-depth insight into CALHM proteins for the first time and provide the founding working model for this unique hemichannel family. These findings will potentially support the development of therapeutic strategies for neurological disorders and diseases mentioned above.